A fundamental goal of pharmacology and medicine is to determine the cellular and molecular mechanisms of ligand-receptor interactions. At the cellular level, drug action is quite often the consequence of non-covalent interactions between therapeutically relevant small organic molecules and high-affinity binding proteins within a specific cell type. These small organic ligands may function as agonists or antagonists of key regulatory events which orchestrate both normal and abnormal cellular functions. For years, the pharmaceutical industry's approach to discovering such ligands has focused on random screenings of thousands of small molecules in specific in vitro and in vivo assays to determine potent lead compounds for their drug discovery efforts. Using these screenings, a lead compound may be found to exert well-defined effects with regard to a function at the cellular level (e.g., inhibition of cytokine production or DNA replication in a particular cancer cell line). However, such results may give little indication as to the mechanism of action at the molecular level (e.g., ligand-protein interaction).
Furthermore, screening for a compound's activity in one cellular function may not reveal its potential for cross-reactivities or its undesired side-effects. Such cross-reactivities and side-effects are often caused, for example, by proteins having closely similar structures to protein(s) under study, or by a single protein fulfilling different functions when expressed in different cell types or when localized to different sub-cellular compartments. Therefore, identifying possible protein targets for a pharmacological agent is challenging but highly desirable. There is an unmet need for a general and efficient method to identify the cellular targets for such pharmacological agents so as to accelerate the search for novel drugs at both the basic and applied levels of research.
Similarly, there is a need for a general approach to identifying a small molecule capable of binding any selected cellular target regardless of the target's biological function. Fowlkes et al. and Broach et al. describe a screening assay for identifying molecules capable of binding cell surface receptors so as to activate a selected signal transduction pathway. These references describe the modification of selected yeast signaling pathways so as to mimic steps in the mammalian signaling pathway. This approach is applicable only to certain signaling pathways and has limited utility for broader applications, such as discovering small molecules that interact with any cellular target. Thus, there is also need for general screening methods to determine the interaction between small molecules and target proteins so as to identify agonists and antagonists that may interfere or compete with the binding of the small molecules for these targets, and to identify new drugs that are capable of specific therapeutic effects in a variety of disease states.
Currently, few efficient methodologies exist for rapidly identifying a biological target such as a protein for a particular small molecule ligand. Existing approaches include the use of affinity chromatography, radio-labeled ligand binding and photoaffinity labeling in combination with protein purification methods to detect and isolate putative target proteins. This is followed by cloning of the gene encoding the target protein based on the peptide sequence of the isolated target. These approaches depend on the abundance of the putative target protein in the sample and are laborious and painstaking.
Crabtree et al. (WO 94/18317) describes a method to activate a target gene in cells comprising (a) the provision of cells containing and capable of expressing (i) at least one DNA construct comprising at least one receptor domain, capable of binding to a selected ligand, fused to a heterologous additional protein capable of initiating a biological process upon exposure of the fusion construct to the ligand, wherein the biological process comprises the expression of the target gene, wherein the ligand is capable of binding to two or more fusion proteins, and wherein the biological process is only initiated upon binding of the ligand to two or more fusion proteins, the two fusion proteins being the same or different, and (ii) the target gene under the expression control of a control element which is transcriptionally responsive to the initiation of said biological process; and (b) exposing said cells to said ligand in an amount effective to result in expression of the reporter gene. Further described are DNA constructs, ligands and kits useful for performing such method. In related documents, Crabtree et al. show these and other embodiments; specifically, Holt et al. describes the synthesis of hybrid ligands for use with the subject methods. The purpose envisaged for these methods and compositions is restricted to the investigation of cellular processes, the regulation of the synthesis of proteins of therapeutic or agricultural importance and the regulation of cellular processes in gene therapy. Nothing therein suggests the use of these methods and compositions to study the interaction of proteins with small molecules, particularly in its application to pharmaceutical research and drug development.
Licitra et al. describes a “three-hybrid screen assay” that implements the basic yeast two-hybrid assay system. (See Fields & Song, Fields et al., Gyuris et al., and Yang et al., for a description of the “two-hybrid assay”) The significant difference is that instead of depending on the interaction between a so-called “bait” and a so-called “prey” protein, the transcription of the reporter gene is conditioned on the proximity of the two proteins, each of which can bind specifically to one of the two moieties of a small hybrid ligand. The small hybrid ligand constitutes the “third” component of the hybrid assay system. In that system, one known moiety of the hybrid ligand will bind to the “bait” protein, while the interaction between the other moiety and the “prey” protein can be exploited to screen for either a protein that can bind a known moiety, or a small moiety (pharmaceutical compound or drug) that can bind a known protein target. Licitra et al. used such an approach to targets for FK506 by fusing a rat glucocorticoid receptor (GR) gene to a lexA-based vector and screened a cDNA library from human leukemia Jurkat cells for proteins to induce reporter gene expression in the presence of a synthetic heterodimeric compound dexamethasone-FK506. Clones expressing a FK506-binding protein (FKBP12) were identified as a mediating partner of the interaction.
Weak affinity between FK506 and FKBP was a limitation of the three-hybrid system disclosed by Licitra et al. The FK506-FKBP interaction utilized by Licitra et al. provided only micromolar affinity. Higher affinity between bait protein and its binding partner is desired for improving system performance. Lin et al. improved upon the Licitra's dexamethasone-FK506 pair by synthesizing a methotrexate-dexamethasone heterodimeric probe molecule, also known as a chemical inducer of dimerization (CID). Lin et al.'s three-hybrid system consisted of a DNA binding protein-dihydrofolate reductase chimera (LexA-DHFR), a transcription activation protein-glucocorticoid binding protein chimera (B42-GR), and a heterodimeric probe molecule consisting of analogs of methotrexate and dexamethasone linked via a biphenyl linker. Dexamethasone and methotrexate have low nanomolar and picomolar affinities to their protein receptors GR and DHFR respectively. This system provided a significant improvement of system performance over Licitra et al.
There is a need to develop and synthesize heterodimeric probe molecules that have improved cell permeability, and toxicity, and are efficient chemical dimerizers of proteins in vivo.